RNA-Immunoprecipitation Sequencing of Actinin-BirA* vs. Control iPSC-CMs
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE144806 | GEO | 2021/08/10
REPOSITORIES: GEO
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Project description:RNA-Immunoprecipitation Sequencing of Actinin-BirA* vs. Control iPSC-CMs
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Project description:The immaturity of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a major limitation for their use in drug screening to identify pro-arrhythmogenic or cardiotoxic molecules. Here, we demonstrate an approach that combines lipid-enriched maturation medium with a high concentration of calcium, nanopatterning of culture surfaces and electrostimulation to generate iPSC-CMs with advanced electrophysiological, structural and metabolic phenotypes. Systematic testing reveals that electrostimulation is the key driver of enhanced mitochondrial development and metabolic maturation and improved electrophysiological properties of iPSC-CMs. Increased calcium concentration strongly promotes electrophysiological maturation, while nanopatterning primarily facilitates sarcomere organisation with minor effect on electrophysiological properties. Transcriptome analysis reveals that activation of HMCES and TFAM targets contributes to mitochondrial development, whereas downregulation of MAPK/PI3K and SRF targets is associated with iPSC-CM polyploidy. These findings provide mechanistic insights into iPSC-CM maturation, paving the way for pharmacological responses that more closely resemble those of adult CMs.
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Project description:RNA-Seq to compare hypoxia responses between human and rhesus macaque iPSC-CMs
2018-09-10 | GSE108676 | GEO
Project description:Heart failure (HF) is a common disease in which the heart cannot meet the metabolic demands of the body. It mostly occurs in individuals 65 years or older. Cardiac transplantation is the best option for patients with advanced HF. High numbers of patient-specific cardiac myocytes (CMs) can be generated from induced pluripotent stem cells (iPSCs) and can possibly be used to treat HF. While some studies found iPSC-CMS can couple efficiently to the damaged heart and restore cardiac contractility, almost all found iPSC-CM transplantation is arrhythmogenic, thus hampering the use of iPSC-CMs for cardiac regeneration. Studies show that iPSC-CM cultures are highly heterogeneous containing atrial-, ventricular- and nodal-like CMs. Furthermore, they have an immature phenotype, resembling more fetal than adult CMs. There is an urgent need to overcome these issues. To this end, a novel and interesting avenue to increase CM maturation consists of modulating their metabolism. Combined with careful engineering and animal models of HF, iPSC-CMs can be assessed for their potential for cardiac regeneration and a cure for HF.
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Project description:N-terminal-acetyltransferases including NAA10 catalyze N-terminal acetylation (Nt-acetylation), an evolutionarily conserved co- and post-translational modification. However, little is known about the role of Nt-acetylation in cardiac homeostasis. To gain insight into cardiac-dependent NAA10 function, we studied a novel NAA10 variant (p.R4S) segregating with QT-prolongation, cardiomyopathy and developmental delay in a large kindred. Here we show that the NAA10R4S variant reduced enzymatic activity, decreased expression levels of NAA10/NAA15 proteins, and destabilized the enzymatic complex NatA. In NAA10R4S/Y-iPSC-CMs, dysregulation of the late sodium and slow rectifying potassium currents caused severe repolarization abnormalities, consistent with clinical QT prolongation. Engineered heart tissues generated from NAA10R4S/Y-iPSC-CMs had significantly decreased contractile force and sarcomeric disorganization, consistent with the pedigree’s cardiomyopathic phenotype. Proteomic studies revealed dysregulation of metabolic pathways and cardiac structural proteins. We identified small molecule and genetic therapies that normalized the phenotype of NAA10R4S/Y-iPSC-CMs. Our study defines novel roles of Nt-acetylation in cardiac regulation and delineates mechanisms underlying QT prolongation, arrhythmia, and cardiomyopathy caused by NAA10 dysfunction.
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Project description:The definition of lncRNAs conservation remain a crucial challenge. In order to identify more promising relevant cardiac ncRNAs, we selected transcripts also present in human cardiac cells. We performed total RNA-seq in human RUES2 and iPSC derived cardiomyocyte at day 15 of differentiation, first for human transcripts identification and secondly for the orthologous selection.
2020-09-07 | GSE143930 | GEO
Project description:High-throughput in vitro drug assays have been impacted by recent advances in human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) technology and by contact-free all-optical systems simultaneously measuring action potentials (APs) and Ca2+ transients (CaTrs). Parallel computational advances have shown that in silico simulations can predict drug effects with high accuracy. We combine these in vitro and in silico technologies and demonstrate the utility of high-throughput experimental data to refine in silico hiPSC-CM populations and to predict and explain drug action mechanisms. Optically obtained hiPSC-CM APs and CaTrs were used from spontaneous activity and under optical pacing in control and drug conditions at multiple doses. An updated version of the Paci2018 model was developed to refine the description of hiPSC-CM spontaneous electrical activity; a population of in silico hiPSC-CMs was constructed and calibrated using simultaneously recorded APs and CaTrs. We tested in silico five drugs (astemizole, dofetilide, ibutilide, bepridil, and diltiazem) and compared the outcomes to in vitro optical recordings. Our simulations showed that physiologically accurate population of models can be obtained by integrating AP and CaTr control records. Thus, constructed population of models correctly predicted the drug effects and occurrence of adverse episodes, even though the population was optimized only based on control data and in vitro drug testing data were not deployed during its calibration. Furthermore, the in silico investigation yielded mechanistic insights; e.g., through simulations, bepridil's more proarrhythmic action in adult cardiomyocytes compared to hiPSC-CMs could be traced to the different expression of ion currents in the two. Therefore, our work 1) supports the utility of all-optical electrophysiology in providing high-content data to refine experimentally calibrated populations of in silico hiPSC-CMs, 2) offers insights into certain limitations when translating results obtained in hiPSC-CMs to humans, and 3) shows the strength of combining high-throughput in vitro and population in silico approaches.
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Project description:This project investigates G3BP1 proximity proteins by constructing a G3BP1-BirA* fusion protein using G3BP1 as a decoy protein and BirA* as a biotin ligase. The changes in G3BP1 proximity proteins were studied in the presence or absence of poly(I:C) stimulation conditions, using poly(I:C) stimulation as a variable. This project investigated the TRIM25 proximity protein by constructing a TRIM25-BirA* fusion protein with TRIM25 as the decoy protein and BirA* as the biotin ligase. The changes of TRIM25 proximity proteins under the conditions of TRIM25 interacting with or without G3BP1 were studied by performing PTFG sequence deletion on TRIM25; the changes in TRIM25 proximity proteins were studied in the presence or absence of poly(I:C) stimulation conditions, using poly(I:C) stimulation as a variable.
2023-07-07 | PXD043603 |
Project description:The ability to differentiate induced-pluripotent stem cells into cardiomyocytes (iPSC-CMs) has opened up novel avenues for potential cardiac therapies. However, iPSC-CMs exhibit a range of somewhat immature functional properties. This study explored the development of the beta-adrenergic receptor (?AR) pathway, which is crucial in regulating contraction and signifying the health and maturity of myocytes. We explored the compartmentation of ?2AR-signalling and phosphodiesterases (PDEs) in caveolae, as functional nanodomains supporting the mature phenotype. Förster Resonance Energy Transfer (FRET) microscopy was used to study the cyclic adenosine monophosphate (cAMP) levels in iPSC-CMs at day 30, 60, and 90 following ?AR subtype-specific stimulation. Subsequently, the PDE2, PDE3, and PDE4 activity was investigated using specific inhibitors. Cells were treated with methyl-?-cyclodextrin (M?CD) to remove cholesterol as a method of decompartmentalising ?2AR. As iPSC-CMs mature with a prolonged culture time, the caveolae density is increased, leading to a reduction in the overall cytoplasmic cAMP signal stimulated through ?2AR (but not ?1AR). Pan-phosphodiesterase inhibition or caveolae depletion leads to an increase in the ?2AR-stimulated cytoplasmic cAMP. Moreover, with time in culture, the increase in the ?AR-dependent cytoplasmic cAMP becomes more sensitive to cholesterol removal. The regulation of the ?2AR response by PDE2 and 4 is similarly increased with the time in culture. We conclude that both the ?2AR and PDEs are restricted to the caveolae nanodomains, and thereby exhibit a tighter spatial restriction over the cAMP signal in late-stage compared to early iPSC-CMs.
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